Genetic Dissection Of Plant-virus Interactions

For a long time of evolution, plants have developed series of mechanisms to defense against viruses including Resistance(R) gene-mediated response, RNA silencing, Lectin protein-mediated response and some other host proteins such as translational initiation factors and endoplasmic reticulum. However, plants viruses also involved a variety of mechanisms to survive from hosts, either through molecular evolution (mutations, recombinations, selection pressure or gene flow), or through encoding proteins (eg, VSRs) to defense against plant immunity. Our study focused on genetic dissection of plant-virus interactions:on one hand, we explored how virus counter-defense plant immunity by using Apple stem pitting virus (ASPV), on the other hand, we study how plant defense against virus by exploring how Arabidopsis defend against Tobacco rattle virus (TRV). Our results are listed as follows:1. Genetic diversity and evolution of ASPV pear isolates in ChinaTo make clear population structure and molecular evolution mechanisms of ASPV from pear in China,451 pear samples from different pear production regions in China were collected, wherein 145 samples were tested ASPV positive. Complete coat protein (CP), triple gene block (TGB) and partial RNA dependent RNA polymerase (RdRP) fragments (P1 and P2) of some of these ASPV isolates were sequenced. In our study, we sequenced 169 CP sequences from 31 ASPV pear isolates,95 TGB sequences from ASPV 44 pear isolates,17 P1 sequences and 14 P2 sequences of RdRP from 3 different isolates. Phylogenetic trees analysis based on these sequenced clones in our study and related sequences on GenBank suggested that:1) ASPV grouping in phylogenetic trees were related to their hosts (apple, pear and Korla pear), no matter which ASPV genes was used; 2) ASPV from pear isolates could be divided into six evolutionary divergent subgroups (A-F) based on their CP sequences, wherein two new subgroups (B and F) were identified in our study; 3) ASPV isolates could be divided into five evolutionary divergent groups based on their TGB sequences, which was extensively studied for the first time. Multiple alignments analysis indicated continuous nucleotides insertions or deletions were exited in CP and RdRP genes of ASPV pear isolates in China. Recombination events were detected in CP, TGB and RdRP sequences in our study. The selection pressure analysis suggested that ASPV CP and TGB genes were under negative selection. Our study suggested insertions or deletions mutations, selection pressure and recombination played important roles in genetic diversity of ASP V pear isolates in China.2. Full length of ASP V pear isolates and ASPV-vsiRNAs analysisFull length of ASPV pear isolate (Pyrus pyrifolia) (Wuhan city, HuBei province) was sequenced in our study, which was named HB-HN1. HB-HN1 was 9,270 nt in length (excluding polyA tail). HB-HN1 shared 72.4%-80.0% similarity with 11 other reported ASPV isolatesat nt leve. However, Results patterns were different if different genes were used. The 5'UTR (Untranslated Region) of ASPV was relatively conserved while the 3' UTR region were highly variable. Phylogenetic tree analysis based on these 12 full length sequences also suggested different ASPV isolates grouping was related to hosts (apple, pear and Korla pear). Results of ASPV-vsiRNAs analysis indicated ASPV positive and negative strand RNAs contributed equally to generated vsiRNAs and these vsiRNAs distributed onto whole ASPV genome. It was interesting to find that 5 teminal of ASPV-vsiRNAs had a C bias.Functions diverisity analysis of proteins encoded by ASPVASPV CP sequences (HB-HN1-3, HB-HN7-18, HB-HN6-8, HB-HN9-3, YN-MRS-17, LN-AP-1) from five pear isolates and 1 apple isolate were selected to express fused CP in Prokaryotes in certain conditions (30?, in LB medium containing 50 mg/L Kan, induced 6 h by 1 mM/L IPTG), in which three were selected to produce recombinant antibody for further analyzing serological reactivity. These three produced polyclonaT antiserums were named PAb-HB-HN9-3, PAb-HB-HN6-8 and PAb-YN-MRS-17. Western blot indicated different extent of serological reactivity between these three antibodies and six fused CP expressed in Prokaryotes. Different isolates of ASPV CP expressed by PVX vector (PVX-ASPV-CP) were found to have VSRs (viral supressor of RNA silencing) founction. Also PVX-ASPV-CP infected Nicotiana occidentalis dispalyed severe symptoms compared to PVX (wt) infected ones. We also confirmed that VSRs function and pathogenicity of ASPV CP were not affected by molecular variation. However, Transmembrane helices (TMHs) strcture of ASPV TGB3 were affected by molecular variation of this gene.Different roles for RNA silencing and P-bodies components in recovery and VIGSA major antiviral mechanism in plants is mediated by RNA silencing, which relies on the cleavage of viral double-stranded RNA into virus-derived small interfering RNAs (vsiRNAs) by DICER-like (DCL) enzymes. Members of the Argonaute (AGO) family of endonucleases then use these vsiRNA as guides to target viral RNA. This can result in a phenomenon known as recovery, whereby the plant silences viral gene expression and recovers from viral symptoms. Endogenous mRNAs can also be targeted by vsiRNAs in a phenomenon known as virus-induced gene silencing (VIGS). Although related to other RNA silencing mechanisms, it has not been established if recovery and VIGS are mediated by the same molecular mechanisms. We have used Tobacco rattle virus carrying a fragment of the PDS gene (TRV-PDS) or expressing GFP (TRV-GFP) as readouts for VIGS and recovery, respectively, in Arabidopsis ago mutants. Our results demonstrate roles for AGO2 and AGO4 in susceptibility to TRV, whereas VIGS of endogenous genes appears to be largely mediated by AGO1. However, recovery appears to be mediated by different components, as all the aforementioned mutants were able to recover from TRV-GFP. TRV RNAs from recovered plants associate less with ribosomes, suggesting that recovery involves translational repression of viral transcripts. Translationally repressed RNAs often accumulate in RNA processing bodies (PBs), where they are eventually processed by decapping enzymes. Consistent with this, we find that viral recovery induces increased PB formation and that a decapping mutant (DCP2) shows increased VIGS and virus RNA accumulation, indicating an important role for PBs in eliminating viral RNA.